The gene expression of collagen type I and decorin was also determined by quantitative RT-PCR (qRT-PCR) using a light cycler instrument 2.0 (Roche Diagnostic, Mannheim, Germany). Target sequences were amplified using LightCycler Primer Sets (Search LC, Heidelberg, Germany) with LightCycler-Fast Start DNA Master SYBR Green I Mix (Roche Applied Science, Mannheim, Germany) following the manufacturer's manual. Reactions were performed in duplicate. At least two independent experiments were performed. For relative quantification of the gene expression, samples were normalized to cyclophilin B.
Lightcycler fast start dna master sybr green 1 mix
Manufactured by Roche
Sourced in Germany, Switzerland
The LightCycler-Fast Start DNA Master SYBR Green I Mix is a ready-to-use reagent for real-time PCR analysis. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, and a thermostable DNA polymerase for amplification.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 2, by Roche (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Lightcycler 2.0 instrument, by Roche (1 mentions)
6 protocols using lightcycler fast start dna master sybr green 1 mix
1
Quantitative RT-PCR Analysis of Collagen I and Decorin
2
Quantitative PCR for Viral Genome Quantification
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Quantitative real-time PCR was used to determine the viral genome copies present in the brains. Every sixth coronal section was pooled from each mouse brain and the genomic DNA was extracted. For cat brains, genomic DNA was extracted from the 100-µm section of each transverse block and the vector genome copies quantified separately at each transverse level. Copies of D2R80A and fMANB vector genome were quantified using LightCycler FastStart DNA Master SYBR Green I mix (Roche) on a StepOne Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and normalized to the GAPDH gene. For each gene assayed, triplicate samples derived from each DNA pool were used for quantification in mouse brains. Duplicate samples at each transverse level of the cat brains were used for quantification. For D2R80A vector genome quantification, the forward primer was designed to bind the GUSB promoter and the reverse primer bound to the D2R80A gene sequence. The primer sequences were as follows: forward: 5′-ACC TCC CGC GCT TTT CTT AG-3′, reverse: 5′-GCT CCA GTT CTG CCT CTC CA-3′. For fMANB vector genome quantification, the primer sequences were as follows: forward: 5′-GCC CAT GGA AAT CCG TAC CT-3′, reverse: 5′-TGC GAT GCA ATT TCC TCA TTT-3′.
3
Quantitative Real-Time PCR Protocol for Gene Expression Analysis
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Q-PCR was performed using the Lightcycler 2.0 instrument (Roche, Basel, Switzerland) with the 4.05 software version. SYBR green detection system was applied as described earlier [67 (link)]. Every of 20 µL reaction mixtures contained 2 µL template cDNA (standard or control), 0.5 µM of specific primer and a previously determined optimum MgCl2 concentration (3.5 µM for one reaction). Light Cycler Fast Start DNA Master SYBR Green I mix (Roche) was used. The real-time PCR program included 10 min denaturation step to activate the Taq DNA Polymerase, followed by a three-step amplification program: denaturation at 95 °C for 10 s, annealing at 56 °C for 5 s, and extension at 72 °C for 10 s. Specificity of reaction products was checked by determination of melting points (0.1 °C/s transition rate). All samples were amplified in triplicate, and hypoxanthine phosphoribosyltransferase (HPRT) gene was used as a reference to normalize obtained results.
The primers were designed using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) (Table 2 ). They were purchased from the Laboratory of DNA Sequencing and Oligonucleotide Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
The primers were designed using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) (
4
Measuring mRNA Expression and Promoter Activity
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mRNA was isolated with the Qiazol Lysis Reagent (Qiagen, Germantown, MD, USA). Total RNA (1 mg) was reverse-transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (Gibco BRL, Waltham, MA, USA) with random hexamer priming. To determine the expressions of the genes, a real-time PCR analysis was performed with the LightCycler-FastStart DNA Master SYBR Green I mix (Roche Diagnostics, Basel, Switzerland) using specific primers for each target gene and glyceraldehyde-3-phosphate dehydrogenase (Table 1 ). The fold changes were calculated using the 2−ΔΔCt method.
The ZKSCAN3 and CHOP promoter sequences were amplified by PCR from human kidney genomic DNA using upstream and downstream primers (Table 1 ). Each promoter fragment was cloned in-frame into a pGL luciferase reporter vector to generate pGL-ZKSCAN3 and pGL-CHOP. The luciferase activity was measured in the samples containing equivalent amounts of proteins using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
The ZKSCAN3 and CHOP promoter sequences were amplified by PCR from human kidney genomic DNA using upstream and downstream primers (
5
Quantitative PCR of gene expression
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qPCR was performed using a Lightcycler 2.0 instrument version 4.05 software (Roche, Basel, Switzerland). Using the primers mentioned above, SYBR green detection system was applied as described earlier [56 (link),57 (link),61 (link),62 (link),63 (link),64 (link)]. Every of 20 μL reaction mixtures contained 4 μL template cDNA, 0.5 µM of specific primer and a previously determined optimum MgCl2 concentration (3.5 M for one reaction). LightCycler Fast Start DNA Master SYBR Green I mix (Roche, Basel, Switzerland) was used. The real-time PCR program included 10 min denaturation step to activate the Taq DNA Polymerase, followed by a three-step amplification program: denaturation at 95 °C for 10 s, annealing at 56 °C for 5 s, and extension at 72 °C for 10 s. Specificity of reaction products was checked by determination of melting points (0.1 °C/s transition rate). Expression of studied genes was related to B2m.
6
Real-Time qPCR Protocol with SYBR Green
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QPCR was performed by means of the Lightcycler 2.0 instrument (ROCHE) with the 4.05 software version. SYBR green detection system was applied as described earlier (19 (link), 25 (link)–27 (link), 35 (link)–37 (link)). Every 20 μl reaction mixtures contains 2 μl template cDNA (standard or control), 0.5 μM of specific primer, and a previously determined optimum MgCl2 concentration (3.5 μM for each reaction). LightCyclerFastStart DNA Master SYBR Green I mix (ROCHE) was used. The real-time PCR program included 10 min denaturation step to activate the Taq DNA Polymerase, followed by a three-step amplification program: denaturation at 95°C for 10 s, annealing at 56°C for 5 s, and extension at 72°C for 10 s. Specificity of reaction products was checked by determination of melting points (0.1°C/s transition rate). Expression of studied genes was related to B2m, only in case of Hcrtr2 to PBGD.
The primers used were designed by Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) (Table1 ). They were purchased from the Laboratory of DNA Sequencing and Oligonucleotide Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
The primers used were designed by Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) (Table
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